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Krajinska ekologija
No full textPričujoča znanstvena monografija je v prvi vrsti namenjena študentom različnih naravoslovnih disciplin, ki se bodo v poklicnem življenju ukvarjali s prostorom, poleg njih želi avtor s to knjigo približati vsebine krajinske ekologije tudi širši javnosti. Zahtevnejše vsebine, modele in tudi zanimivosti bo bralec našel v vsakem poglavju v rubriki »Podrobno«. Vsi teoretični primeri so opremljeni s slikovnimi izseki iz slovenskih krajin. Slovenija je gozdnata država, zato je razumljivo, da v praktičnih primerih avtor v večji meri obravnava prav mesto in vlogo gozda v prostoru v luči krajinsko ekoloških spoznanj
Vloga androgenih hormonov in 11-oksiandrogenih metabolitov pri raku endometrija in raku jajčnikov
No full textBackground: Endometrial cancer (EC) and ovarian cancer (OC) are the sixth and seventh most diagnosed cancers in women worldwide, with incidence rates rising due to demographic changes. Both cancers mainly affect postmenopausal women, with a median diagnosis age in the early to mid-sixties. EC generally has better survival rates, whereas OC, especially its high-grade serous subtype (HGSOC), is often diagnosed at an advanced stage, resulting in poor survival and frequent chemoresistance. Currently, there is an unmet need for accurate, cost-effective triage tools, less invasive diagnostics, reliable prognostic biomarkers, and new therapeutic targets for both cancers. Steroid hormones have been poorly studied for their role in the pathophysiology of both EC and OC. They are also gaining new attention in cancer research for their role in modulating anti-tumor immunity and chemoresistance. Others and our group have shown that both ECs and HGSOCs express steroid-metabolizing enzymes and receptors, supporting intra-tumoral steroid interconversion that may influence local hormone signaling and thereby impact tumor cell behavior. In this doctoral dissertation, we focused on a specific class of steroid hormones, namely androgens and their 11-oxygenated derivatives, i.e., 11-oxyandrogens. We systematically investigated: (i) the local metabolism of classic androgen and 11-oxyandrogen precursors in well-characterized cell models(ii) the transcriptomic and metabolomic effects of potent androgens and 11-oxyandrogens on cell models, along with the prognostic relevance of steroid-metabolizing enzymes and steroid receptors in tumor tissuesand (iii) the diagnostic potential of these steroids in distinguishing EC and OC from benign uterine conditions and non-malignant adnexal masses, respectively.
Methods: We used four EC and six HGSOC cell lines, along with immortalized control cell lines derived from normal endometrial and ovarian surface epithelium. To investigate local androgen metabolism, we performed quantitative gene expression analysis of key enzymes in androgen and 11-oxyandrogen metabolism and incubated cell lines with physiologically relevant concentrations of classical androgen precursors (dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and androstenedione (A4)) as well as 11-oxyandrogen precursors (11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4)). The resulting steroid metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative gene expression analysis of key enzymes involved in classic and 11-oxyandrogen metabolism was performed using qPCR, and the metabolic profiles were interpreted in the context of enzyme expression levels. To assess the functional effects of androgens, selected EC and HGSOC cell lines were treated with potent classic and 11-oxyandrogens, followed by untargeted transcriptomic analysis via RNA sequencing and metabolomic profiling using nuclear magnetic resonance (NMR). Additionally, publicly available clinical datasets were analyzed to evaluate the expression of key steroid-metabolizing enzymes and steroid receptors in EC and HGSOC tumors, along with their associations with patient survival. Finally, two prospective clinical studies were conducted to evaluate the diagnostic potential of circulating steroids in EC and OC. Serum steroid levels were quantified by LC-MS/MS, and diagnostic accuracy was assessed using machine learning.
Results: (i) In both EC and HGSOC cell models, classic androgen precursors did not support local production of 11-oxyandrogens, attributable to the observed absence of CYP11B1 expression across all cell lines. Furthermore, DHEAS and DHEA served as limited sources of bioactive androgens, testosterone (T) and 5α-dihydrotestosterone (DHT), primarily due to low HSD3B1/2 expression, likely diverting DHEA toward alternative metabolites such as 5α-androstenediol via reductive HSD17B enzymes or oxygenated derivatives through CYP3B isoforms. Among EC cell lines, the low-grade model RL95-2 exhibited the highest capacity to metabolize DHEAS and DHEA into T. In HGSOC, the primary tumor cell line Caov-3 and the non-cancerous control line HIO-80 showed the greatest T production from these precursors. Similarly, A4 was a poor substrate for bioactive androgen synthesis in both cancer types, suggesting that it is preferentially converted to 5α-reduced metabolites via SRD5A isoforms, which were expressed by both EC and HGSOC cells. In contrast, 11-oxyandrogen precursors, 11OHA4 and 11KA4, were efficiently converted into the potent androgen receptor (AR) agonist 11-ketotestosterone (11KT) in selected cell lines. This conversion, catalyzed by the AKR1C3 enzyme, was most pronounced in low-grade EC models (Ishikawa, HEC-1-A, RL95-2) and chemo-sensitive HGSOC lines (OVSAHO, Kuramochi). Notably, the amount of 11KT formed from 11-oxyandrogen precursors was several-fold higher than T produced from classic precursors, underscoring intra-tumoral 11-oxyandrogen metabolism as significant sources of androgen receptor (AR)-activating metabolites in low-grade ECs and chemo-sensitive HGSOCs.
(ii) In EC cell models, treatment with DHT and 11-keto-dihydrotestosterone (11KDHT) induced modest transcriptomic changes after 48 hours of incubation. In AR-expressing Ishikawa cells, androgen exposure upregulated genes such as MYO1D and LAMC3, whereas no significant transcriptomic effects were observed in RL95-2 cells, which express low AR levels. In contrast, the AR-positive HGSOC model OVSAHO exhibited pronounced transcriptomic responses to potent androgens (T, DHT) and 11-oxyandrogens (11KT, 11KDHT) following 72 hours of incubation. These responses included activation of stress response and cell cycle-related pathways. At the metabolomic level, both EC and HGSOC cell models exhibited mild intracellular alterations following 72 hours of incubation with potent androgens and 11-oxyandrogens. Specifically, in the low-grade EC model Ishikawa, DHT and 11KDHT reduced intracellular levels of amino acids and lactic acid, while in the HGSOC model OVSAHO, 11KDHT lowered intracellular amino acid and glutathione levels. Analysis of tumor tissues revealed that expression patterns of AR and steroid-metabolizing enzymes varied by tumor type and clinical characteristics. In EC, higher intra-tumoral expression of HSD11B2, SRD5A2, and AR was associated with improved survival, whereas elevated SRD5A1 expression correlated with poorer outcomes. Similarly, in HGSOC, increased expression of HSD11B2, HSD17B2, and AR was linked to better survival, while higher levels of PAPSS1, H6PD, and HSD17B4 were associated with worse prognosis.
(iii) In patients with EC (n = 62), preoperative serum levels of classic androgens (A4, T), 11-oxyandrogens (11OHA4, 11OHT), and glucocorticoids (17α-hydroxyprogesterone, 11-deoxycortisol) were elevated compared to controls with benign uterine conditions (n = 70). Conversely, patients with primary or recurrent OC (n = 43) showed reduced preoperative serum levels of T and 11-oxyandrogens (11OHT, 11KT) relative to controls with non-malignant adnexal masses (n = 56). Individually, steroid hormones had limited diagnostic accuracy for both EC and OC (AUC < 0.7). However, in EC, a logistic regression model combining the androgen pool (DHEA, A4, T), tumor biomarkers (CA125, HE4), and clinical parameters (BMI, parity) achieved an AUC of 0.87 (sensitivity 74.7%, specificity 79.1%), significantly outperforming CA125, HE4, or their combination alone in differentiating EC from benign uterine conditions. For OC, two models integrating 11-oxyandrogens, age, and either CA125 or HE4 reached an AUC of 0.91, with sensitivities of 88.9% and 94.4% and specificities of 82.0% and 77.3%, respectively, surpassing CA125, HE4, and the ROMA index in differentiating OC from non-malignant adnexal masses. These findings emphasize the added diagnostic value of steroid hormone profiling in the preoperative evaluation of gynecologic tumors, however validation in independent cohorts is needed.
Conclusions: This doctoral dissertation provides new insights into (i) local steroid metabolism in EC and HGSOC cell models(ii) transcriptomic and metabolomic effects of androgen signaling in EC and HGSOC cell modelsand (iii) diagnostic potential of circulating steroids in distinguishing EC and OC from their respective control counterparts. We identified distinct patterns of 11-oxyandrogen metabolism across tumor types and clinical subgroups, between low- and high-grade EC, between EC tumors and normal endometrium, and between chemo-sensitive and chemo-resistant HGSOC models, as well as between HGSOC tumors and normal ovarian epithelium. Functionally, androgen and 11-oxyandrogen exposure triggered significant transcriptomic changes in AR-expressing HGSOC cells, particularly in stress response and cell cycle pathways. In tumor tissues, we identified prognostic biomarker candidates, including steroid-metabolizing enzymes and AR, that could improve risk stratification and support personalized clinical management. Importantly, we also uncovered, for the first time, systemic alterations in circulating 11-oxyandrogens in patients with EC and OC compared to their respective control groups. By integrating steroid profiles with traditional tumor markers and clinical parameters we developed novel diagnostic models for EC and OC, which significantly outperformed tumor biomarkers CA-125 and HE4. To our knowledge, these are the first steroid-protein-based models for these cancers. If validated in larger, independent cohorts, they hold promise to improve cancer detection and ultimately improve clinical outcomes.Ozadje: Rak endometrija (EC) in rak jajčnikov (OC) sta šesti oziroma sedmi najpogosteje diagnosticirani obliki raka pri ženskah po svetu, pri čemer incidenca narašča zaradi demografskih sprememb. Oba raka najpogosteje prizadeneta ženske po menopavzi, v zgodnjih do srednjih šestdesetih letih. EC praviloma omogoča boljše preživetje, medtem ko OC, zlasti serozni karcinom visokega gradusa (HGSOC), pogosto diagnosticirajo v napredovalem stadiju, kar vodi v slabši izid in pogosto kemorezistenco. Trenutno obstaja velika potreba po natančnejših, cenovno sprejemljivih orodjih triaže, zgodnjih in manj invazivnih diagnostičnih metodah, zanesljivih prognostičnih označevalcih ter novih terapevtskih tarčah za obe vrsti raka. Vloga steroidnih hormonov v patofiziologiji EC in OC je bila doslej slabo raziskana, vendar steroidni hormoni pridobivajo vse več pozornosti zaradi vpliva na protitumorsko imunost in kemorezistenco. Naša in druge raziskovalne skupine so pokazale, da tako EC kot HGSOC izražata gene encimov, vključenih v sintezo in metabolizem steroidov, in njihove receptorje, kar omogoča sintezo in delovanje v tumorskih celicah, ki lahko vplivata na lokalni prenos signala hormonov in posledično na signalne poti in odziv tumorskih celic. V doktorski disertaciji smo se osredotočili na skupino steroidnih hormonov – androgene in njihove 11-oksigenirane derivate, tj. 11-oksiandrogene. Sistematično smo preučili: (i) lokalni metabolizem prekurzorjev androgenov in 11-oksiandrogenov v dobro okarakteriziranih celičnih modelih(ii) transkriptomske in metabolomske učinke aktivnih androgenov in 11-oksiandrogenov v celičnih modelih, skupaj z napovedno vrednostjo encimov, vključenih v metabolizem steroidov, in steroidnih receptorjev v tumorskem tkivuter (iii) diagnostični potencial teh steroidov pri razlikovanju EC in OC od benignih patologij endometrija in jajčnikov.
Metode: Uporabili smo štiri celične linije EC in šest celičnih linij HGSOC ter kontrolne linije, pridobljene iz normalnega endometrijskega epitela in površinskega epitela jajčnikov. Za preučevanje lokalnega metabolizma androgenov smo izvedli kvantitativno analizo izraženosti genov ključnih encimov, vključenih v metabolizem klasičnih androgenov, in 11-oksiandrogenov, ter inkubirali celične linije s fiziološko relevantnimi koncentracijami prekurzorjev androgenov (dehidroepiandrosteron sulfat (DHEAS), dehidroepiandrosteron (DHEA) in androstendion (A4)) ter prekurzorjev 11-oksiandrogenov (11β-hidroksiandrostendion (11OHA4) in 11-ketoandrostendion (11KA4)). Nastale steroide smo identificirali in kvantificirali s tekočinsko kromatografijo sklopljeno z masno spektrometrijo (LC-MS/MS). Za funkcionalno oceno učinkov androgenov smo izbrane celične linije EC in HGSOC inkubirali z biološko aktivnimi klasičnimi in 11-oksiandrogeni, nato pa izvedli netarčno transkriptomsko analizo (RNA sekvenciranje) ter metabolomsko analizo z jedrsko magnetno resonanco (NMR). Poleg tega smo analizirali javno dostopne baze podatkov, da bi ocenili izražanje genov ključnih encimov, vključenih v metabolizem steroidov, in steroidnih receptorjev v tumorjih EC in HGSOC ter njihovo povezavo s preživetjem bolnic. Na koncu smo izvedli dve prospektivni klinični študiji za oceno diagnostičnega potenciala sistemskih steroidov pri EC in OC. Ravni serumskih steroidov smo določili z LC-MS/MS, njihov diagnostični potencial pa ocenili z metodami strojnega učenja.
Rezultati: (i) Pri obeh vrstah raka, tako EC kot HGSOC, prekurzorji androgenov niso omogočali tvorbe 11-oksiandrogenov zaradi odsotnosti izražanja gena CYP11B1 v vseh celičnih linijah. Poleg tega sta bila DHEAS in DHEA omejena vira bioaktivnih androgenov, testosterona (T) in 5α-dihidrotestosterona (DHT), in sicer predvsem zaradi nizke izraženosti genov HSD3B1/2, kar je najverjetneje povzročilo pretvorbo DHEA v alternativne metabolite, kot je 5α-androstendiol (prek reduktivnih HSD17B encimov), ali oksigenirane derivate (prek CYP3B izooblik). Med celičnimi linijami je model EC nizkega gradusa RL95-2 izkazoval najvišjo sposobnost pretvorbe DHEAS in DHEA v T. Pri HGSOC sta celična linija primarnega HGSOC Caov-3 in kontrolna linija HIO-80 tvorili največ T iz teh prekurzorjev. Tudi A4 je bil slab substrat za sintezo bioaktivnih androgenov pri obeh vrstah raka, kar nakazuje, da se verjetno pretvarja v alternativne metabolite, vključno s 5α-reduciranimi metaboliti preko izooblik SRD5A, ki so jih izražale tako celice EC kot HGSOC. Nasprotno so se prekurzorji 11-oksiandrogenov (11OHA4 in 11KA4) v izbranih celičnih linijah učinkovito pretvorili v 11-ketotestosteron (11KT), ki je pomemben agonist receptorja za androgene (AR). Ta pretvorba je bila najizrazitejša v modelih EC nizkega gradusa (Ishikawa, HEC-1-A, RL95-2) in kemosenzitivnih linijah HGSOC (OVSAHO, Kuramochi).
Pomembno je, da je bila količina 11KT, ki je nastal iz prekurzorjev 11-oksiandrogenov, pri modelih EC nizkega gradusa in kemosenzitivnega HGSOC večkrat višja kot količina T iz klasičnih prekurzorjev, kar poudarja pomen sinteze 11-oksiandrogenov v tkivu tumorja kot ključnega vira agonistov AR v teh tumorskih modelih.
(ii) V celičnih modelih EC je 48-urna inkubacija z DHT in 11-keto-DHT (11KDHT) na ravni transkriptoma povzročila zmerne spremembe. V celicah Ishikawa, ki izražajo AR, je izpostavljenost androgenom povečala izražanje genov, kot je MYO1D, medtem ko pri RL95-2 celicah z nizko izraženim AR niso bile zaznane statistično značilne spremembe. Model HGSOC OVSAHO, ki prav tako izraža AR, je po 72-urni inkubaciji na ravni transkriptoma izkazal izrazite odzive na androgene (T, DHT) in 11-oksiandrogene (11KT, 11KDHT), vključno z aktivacijo poti, vpletenih v odziv na stres in uravnavanje celičnega cikla. Na metabolomski ravni sta oba modela EC in HGSOC pokazala manjše spremembe po inkubaciji z androgeni. V modelu EC nizkega gradusa Ishikawa sta DHT in 11KDHT zmanjšala raven aminokislin in mlečne kisline, medtem ko je pri modelu HGSOC OVSAHO 11KDHT znižal raven aminokislin in glutationa. Analiza tumorskih tkiv je pokazala, da se izražanje gena AR in genov, ki kodirajo encime, vključenih v metabolizem steroidov, razlikuje glede na tip tumorja in klinične značilnosti. Pri EC je bila višja izraženost genov HSD11B2, SRD5A2 in AR povezana z boljšim preživetjem, medtem ko je bila višja izraženost gena SRD5A1 povezana s slabšim izidom. Podobno je bilo pri HGSOC večje izražanje genov HSD11B2, HSD17B2 in AR povezano z boljšim preživetjem, medtem ko je bila višja izraženost genov PAPSS1, H6PD in HSD17B4 povezana s slabšo prognozo.
(iii) Pri bolnicah z EC (n = 62) so bile predoperativne ravni klasičnih androgenov (A4, T), 11-oksiandrogenov (11OHA4, 11OHT) in glukokortikoidov (17α-hidroksiprogesteron, 11-deoksikortizol) povišane v primerjavi z bolnicami z benignimi boleznimi maternice (n = 70). Nasprotno pa so imele bolnice s primarnim ali ponavljajočim se OC (n = 43) znižane predoperativne serumske ravni T in 11-oksiandrogenov (11OHT, 11KT) v primerjavi z bolnicami z benignimi adneksalnimi masami (n = 56). Posamično so imeli steroidni hormoni omejeno diagnostično moč za EC in OC (AUC < 0,7). Vendar pa je za EC logistični regresijski model, ki je združeval androgene (DHEA, A4, T), tumorske biooznačevalce (CA-125, HE4) in klinične parametre (indeks telesne mase (ITM), pariteto), dosegel AUC 0,87 (občutljivost 74,7 %, specifičnost 79,1 %) in s tem pomembno presegel diagnostično moč CA-125, HE4 ali njune kombinacije. Za OC sta dva modela, ki sta vključevala 11-oksiandrogene, starost in bodisi CA-125 bodisi HE4, dosegla AUC 0,91, z občutljivostmi 88,9 % in 94,4 % ter specifičnostmi 82,0 % in 77,3 %, kar je preseglo diagnostično učinkovitost CA-125, HE4 in ROMA indeksa pri razlikovanju OC od benignih adneksalnih mas. Naši rezultati poudarjajo dodatno diagnostično vrednost profiliranja steroidnih hormonov pri predoperativni oceni ginekoloških tumorjev, vendar je potrebna validacija na neodvisnih kohortah.
Zaključki: Doktorska disertacija prinaša nova spoznanja o (i) lokalnem metabolizmu steroidov v celičnih modelih EC in HGSOC(ii) vplivu androgenov na transkriptom in metabolomter (iii) diagnostičnem potencialu sistemskih steroidov pri razlikovanju EC in OC od kontrolnih skupin. Ugotovili smo različne vzorce metabolizma 11-oksiandrogenov med modeli EC nizkega in visokega gradusa, med modeli EC in normalnim endometrijskim epitelijem, med kemosenzitivnimi in kemorezistentnimi modeli HGSOC ter med modeli HGSOC in normalnim ovarijskim epitelijem. Funkcionalno je izpostavljenost androgenom in 11-oksiandrogenom sprožila pomembne spremembe na ravni transkriptoma v AR-pozitivnem modelu HGSOC, zlasti v signalnih poteh odziva na stres in celičnega cikla. V tumorskem tkivu smo identificirali kandidate za prognostične označevalce, vključno z encimi za sintezo in metabolizem steroidov, ter AR, ki bi lahko podprli personalizirano klinično obravnavo. Pomembno je, da smo prvi ugotovili sistemske spremembe v 11-oksiandrogenih pri bolnicah z EC in OC v primerjavi z bolnicami z benignimi patologijami. Z integracijo steroidnih profilov s tradicionalnimi tumorskimi biooznačevalci in kliničnimi parametri smo razvili nove modele za neinvazivno diagnostiko EC in OC, ki pomembno presegajo trenutno uporabljene serumske biooznačevalce, CA-125 in HE4. Če bodo validirani v večjih, neodvisnih kohortah, imajo ti modeli potencial za izboljšano in neinvazivno odkrivanje teh rakov ter posledično izboljšanje kliničnih izidov
Design, synthesis and evaluation of heteroaromatic dual inhibitors of butyrylcholinesterase and p38α mitogen-activated protein kinase
No full textS staranjem populacije postaja Alzheimerjeva bolezen (AB), najpogostejša nevrodegenerativna motnja in oblika demence, vse večji izziv za bolnike, svojce in zdravstvene sisteme. Pri postopnem upadanju holinergičnega prenosa pri bolnikih z AB ima pomembno vlogo butirilholin esteraza (BChE). Z mitogenom aktivirana protein kinaza p38α (p38α MAPK), ki je vključena v proces nevrovnetja, hiperfosforilacije proteina tau in v patofiziologijo amiloidnih plakov, pa je validirana tarča za upočasnjevanja nevrodegeneracije pri tej bolezni.
V tej doktorski disertaciji smo računalniško načrtovali, sintetizirali in ovrednotili prvi strukturni razred dvojnih zaviralcev BChE/p38α MAPK kot možen pristop za zdravljenje AB. Z zamenjavo fenoksi skupine v spojini zadetku z benzilno smo dosegli π-π interakcijo s Trp430 v holin-vezavnem žepu BChE in ustrezno vezavo v hidrofobno regijo I p38α MAPK, kar je privedlo do spojin z nanomolarnimi jakostmi zaviranja obeh tarčnih encimov. Najobetavnejši spojini sta bili selektivni za p38α MAPK v panelu 103 kinaz in učinkoviti v in vivo modelih kognitivnih motenj.
Z racionalnim načrtovanjem na podlagi kristalnih struktur prej omenjenih spojin v aktivnih mestih obeh encimov smo uspešno pripravili serijo karbamatnih plejotropnih predzdravil, ki po psevdo-ireverzibilni vezavi na Ser198 BChE sprostijo alkohol, ki nato kompetitivno zavira p38α MAPK.
V želji po razvoju močnejših zaviralcev p38α MAPK, a ohranjeni visoki selektivnosti napram ostalim kinazam, smo načrtovali še eno serijo karbamatnih dvojnih zaviralcev BChE/p38α MAPK, ki se kovalentno vežejo v aktivno mesto BChE in so tudi alosterični zaviralci p38α MAPK. Z uvedbo karbamatne skupine smo pripravili spojine z nizko nanomolarno afiniteto za BChE in prokognitivnimi učinki v in vivo modelu kognitivnega upada. Ta sprememba strukture je povzročila izgubo pomembnih interakcij v alosteričnem vezavnem mestu p38α MAPK in s tem prenizko aktivnost za ovrednotenje kinazne selektivnosti.With an aging population, Alzheimer’s disease (AD) — the most common neurodegenerative disorder and form of dementia — increasingly challenges patients, families, and healthcare systems. Butyrylcholinesterase (BChE) plays a key role in the progressive decline of cholinergic transmission observed in AD. Mitogen-activated protein kinase p38α (p38α MAPK), on the other hand is emerging as a validated target in neurodegeneration, tau hyperphosphorylation, and amyloid plaque pathology.
In this doctoral dissertation, we computationally designed, synthesized, and evaluated first-in-class dual inhibitors of hBChE/p38α MAPK as potential anti-AD drugs. Replacing the phenoxy group of the hit compound with a benzyl group enabled π-π stacking with Trp430 of the BChE choline-binding pocket and favourable binding within p38α MAPK’s hydrophobic region I, achieving nanomolar inhibitory potencies on both targets. The two lead compounds showed selectivity for p38α MAPK in a panel of 103 kinases and were effective in vivo in two models of cognitive decline.
Subsequently, using rational design based on the crystal structures of the complexes of these compounds with the target enzymes, we developed carbamate-based pleiotropic prodrugs of p38α MAPK inhibitors. These prodrugs are activated via pseudo-irreversible covalent binding to Ser198 of BChE, releasing an alcohol that competitively inhibits p38α MAPK.
To enhance the affinity for p38α MAPK while retaining high selectivity over other kinases, we designed another series of dual carbamate inhibitors that covalently bind to BChE and act as allosteric p38α MAPK inhibitors. Introduction of the carbamate group produced compounds with low nanomolar affinity for BChE and procognitive effects in vivo, but also caused a loss of important interactions in the allosteric site of p38α MAPK and thus insufficient inhibitory potency for kinase selectivity evaluation
Vloga z obsevanjem povzročenih sprememb tumorskega žilja na infiltracijo imunskih celic pri raku debelega črevesa
No full textColon cancer is the second most prevalent cancer in women and the third in men. The conventional therapies for treating colon cancer include surgery, chemotherapy, and radiotherapy (RT)however, they are often ineffective. The main mode of action of RT is the killing of proliferating cancer cells by causing double-strand DNA breaks. Besides, it leads to a release of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as damage-associated molecular patterns (DAMPs) that activate dendritic cells to present tumor antigens to naive T lymphocytes. Consequently, naive T lymphocytes differentiate into effector T lymphocytes, primarily cytotoxic CD8+ T cells and helper CD4+ T cells, which begin to secret chemokines and cytokines, triggering an immune response. Tumors consist of cancer cells and tumor microenvironment (TME). An important part of TME are tumor blood vessels, which are required for tumor progression due to supplying them with nutrients and oxygen. In contrast to normal vasculature, tumor vasculature is not fully functional, as it is immature, disorganized, and inconsistent, which results in inadequate blood flow and the formation of hypoxic microregions, resistant to RT. Importantly, irradiation (IR) does not only affect cancer cells but also TME, including tumor vasculature. However, the response of the TME, including tumor vasculature is poorly understood. The effect of IR on tumor endothelium is time- and dose-dependent. IR can cause apoptosis of endothelial cells (ECs), with proliferating ECs being more susceptible. However, there is little evidence of vascular destruction with single doses of radiation below ~15 Gy or with daily fractionated doses. In addition to anti-angiogenic agents, IR alone can also act as a vascular normalization therapy. Besides the increased sensitivity of tumor cells to IR due to reduced levels of hypoxia, vascular normalization can also improve access of immune cells to previously non-perfused regions. Additionally, doses higher than 2 Gy may lead to endothelial activation, during which ECs switch from anti-inflammatory into pro-inflammatory phenotype. The underlying mechanism for the endothelial activation after IR remains unknown. The activated endothelium is characterized by the overexpression of adhesion molecules and pro-inflammatory chemokines and cytokines, contributing to the infiltration of immune cells. Recently, a subtype of blood vessels termed tumor associated high endothelial venules (TA-HEVs) was shown to represent the entry site for lymphocytes into tumors. However, the exact crosstalk between IR-activated tumor ECs, whether they are connected to TA-HEVs, and different populations of immune cells is yet to be elucidated. Therefore, the aim of the doctoral dissertation was to improve the understanding of the response of tumor blood vessels to RT by determining how the IR-induced changes to the vascular structure and endothelial activation are connected to the homing and extravasation of immune cells into the TME.
In the first Work package, we investigated the effects of IR on the vascular network and endothelial activation in vitro. We assessed the response of murine EC lines bEnd.3, 2H11, and SVEC4-10 and human endothelial cell lines HUVEC, EA.hy926, Hulec-5a grown as monolayers to different IR doses and time after the exposure to IR. In the cell proliferation and survival experiments, cells were subjected to 2-10 Gy of IR one day after seeding. Immediately before IR treatment (0 h) and at different timepoints (24, 48, 72, and 96 h) after IR, cells were incubated in phosphate-buffered saline (PBS) solution containing Hoechst 33342 and Propidium Iodide (PI). The number of live cells (Hoechst 33342 positive nuclei) and the number of dead cells (PI positive nuclei) were quantified to calculate relative change of proliferation, number of normalized dead cells and doubling time. To evaluate IR-induced changes in the HUVEC transcriptome, RNA sequencing analysis was performed on HUVECs irradiated with either 2 or 5 Gy and compared to non-irradiated samples. Total RNA of non-irradiated and irradiated samples was extracted at 24 h or 72 h after IR. Transcriptome sequencing was conducted by Novogene Company LTD, using the Illumina platform. RNA sequencing analysis was performed using R software, focusing on significantly changed genes and pathways related to endothelial activation and immune response. In the next step, we established a vasculature-on-a-chip model, which enabled us to examine IR-induced changes in a physiologically relevant 3D microvascular environment. First, we attempted to reproduce a microfluidic chip protocol based on published literature the Chen et al. model. To establish a model capable of forming perfusable vascular networks, we tested the murine EC lines bEND.3, 2H11, and SVEC4-10, and the human EC lines EA.hy926, Hulec-5a and HUVEC. In addition, we optimized the EC seeding density, the EC:fibroblast ratio, the concentrations of thrombin and fibrinogen, and the introduction of interstitial flow. However, due to reproducibility issues, we adopted the OrganoPlate® microfluidic chips (Mimetas). Once established, the microvascular networks were irradiated with 2 or 5 Gy, based on prior IR dose-response results. To assess vascular permeability and integrity, Dextran dye was perfused into the microchips at 24 and 72 h after IR. Endothelial activation was evaluated by immunofluorescence staining for von Willebrand factor (VWF), Intercellular Adhesion Molecule 1 (ICAM-1), Interferon Regulatory Factor 9 (IRF9), and Interferon α/β (IFNα/β). Imaging was conducted with ZEISS ELYRA 7 Lattice SIM system. To assess interaction between irradiated endothelium and immune cells, fluorescently labeled CD8+ T cells, isolated from human buffy coat, were perfused through the microchips at 24 h and 72 h after IR. Imaging was conducted using Axio Observer 7 equipped with Colibri 7 LED light source UV and a Hamamatsu Orca Flash camera. The acquired images were processed and visualized using Imaris software (version 9.9.1). In the second Work package, we determined the level of vascular normalization and endothelial activation in vivo (Permit No. U34401-36/2020/7 and U34401-19/2024/9). We first irradiated MC38 and CT26 colon carcinoma tumors subcutaneously grown in C57Bl/6 or Balb/c mice, respectively, with either a single dose of 15 Gy or fractionated dose of 5 x 5 Gy and determined the response to RT. IR was conducted with a Gulmay CP225 X-RAY generator with 0.55 mm copper and 1.8 mm aluminum filtering, operated at 200 kV, 9.2 mA, with a dose rate of 1.92 Gy/min. To assess the effect of IR on the induction of endothelial activation and the formation of TA-HEVs, frozen sections of CT26 colon carcinomas excised at 24 h, 48 h, and 7 days after IR were immunofluorescently stained for the endothelial activation markers VWF and ICAM-1, the TA-HEV marker Peripheral Node Addressin Antibody (MECA-79), and markers associated with immune response IRF9, Cluster of differentiation 8 (CD8), and Cluster of differentiation 4 (CD4). The Cluster of differentiation 31 (CD31) was used to identify vasculature. Cell proliferation and hypoxia were evaluated by incorporation of thymidine analogue EdU and nitroimidazole derivative EF5 staining, respectively. Immunofluorescently stained tumor sections were imaged using Axio Observer 7 equipped with Colibri 7 LED light source UV and a Hamamatsu Orca Flash camera and analyzed using Imaris software (version 9.9.1). To investigate the transcriptional changes following IR, we used the 10x Genomics Visium Spatial Transcriptomics technology. Formalin-Fixed Paraffin-Embedded (FFPE) control and irradiated CT26 tumors excised at 48 h and 7 days after completion of fractionated 5 x 5 Gy RT, as well as tumor biopsies from three patients (Permit No. 0120-394/2023/3) obtained before neoadjuvant 5 x 5 Gy RT and samples from tumor resection after RT were analyzed. FFPE sections were mounted onto Visium Spatial Transcriptomics slides with capture probes, H&E stained, imaged, and processed for library preparation. Sequencing was conducted by Novogene, and the transcriptomic data were analyzed using using 10X Genomics SpaceRanger analysis pipeline (version 4.0) and a visualization software LoupeBrowser (version 8.0). We focused on IR-induced vascular changes associated with endothelial activation, immune cell infiltration, and TA-HEV formation. Immunohistochemistry on FFPE sections was performed for validation of the Spatial transcriptomics results using antibodies against CD31, CD4, CD8, IRF9, and Granzyme B. In the last Work package, the homing of immune cells following IR was investigated using the dorsal skinfold window chamber model, which enables real-time visualization of the tumor vasculature over time. Two symmetrical titanium frames, were surgically implanted into the dorsal skin of VE-TOM transgenic mice expressing fluorescent protein tdTomato in ECs. A circular window (~10 mm in diameter) was created by removing one skin layer, exposing the subcutaneous tissue and vasculature. MC38 colon carcinoma cells were injected into the remaining subcutaneous tissue of VE-TOM mice. Once the tumors reached a visible size of ~4 mm in diameter, they were irradiated with a single dose of 15 Gy using a specially designed collimator that allows IR of only the tissue within a 6 × 6 mm square inside the window chamber. Tumors were irradiated using the Gulmay 225 X-ray system with 0.55 mm Cu and 1.8 mm Al filtering with IR dose rate if 0.97 Gy/min. Vascular and immune cells\u27 dynamics were monitored using intravital microscopy. A fluorescently labeled anti-MECA-79 antibody was injected intravenously (i.v.) to visualize the TA-HEVs. Further, fluorescently labelled CD8+ T cells isolated from mouse spleen were injected i.v. before imaging to determine their interaction with ECs. Imaging was conducted under isoflurane inhalation anesthesia using a Zeiss LSM 800 confocal microscope. The acquired 3D images were processed and visualized using Imaris software (version 9.9.1). For data analysis, visualization, and statistics, GraphPad, R software, and 10x Genomics software were used. Statistical methods were selected individually for each type of data. Statistical significance was determined using One-way, Two-way and mixed-design analysis of variance (ANOVA), linear mixed model (LMM), Wald test, and t-test. Not normally distributed data were analyzed with non-parametric tests (Wilcoxon test, Mann-Whitney test, and Kruskal-Wallis test).IR of murine bEnd.3, 2H11, and SVEC4-10 and human HUVEC, EA.hy926, and Hulec5a ECs with single doses of 2-10 Gy resulted in a dose-dependent reduction of proliferation and increase in EC death. Among the murine EC lines, bEnd.3 was the most radiosensitive, followed by 2H11 and SVEC4-10. In the bEnd.3 EC line, a reduced proliferation was already determined after the 2 Gy single dose IRwhereas, in the 2H11 and SVCE4-10 EC lines reduced proliferation was determined after 4 Gy. The percentage of dead cells confirmed that the most radiosensitive EC line was bEnd.3, as the percentage of dead cells was statistically significantly increased in all timepoints after IR with 4–10 Gy, which was not the case in 2H11 and SVEC4-10. In all murine EC lines tested, the doubling time was significantly prolonged after IR with 10 Gyhowever, in bEnd.3 cells, a significantly longer doubling time was also observed after IR with 6 and 8 Gy. Among all tested human EC lines, HUVECs were the most radiosensitive, while the radiosensitivity of EA.hy926 and Hulec5a was lower but comparable. A statistically significant decrease in proliferation of HUVECs and Hulec-5a, compared to control, was observed regardless of the dose and timepoint, whereas EA.hy926 did not show significance at 24 h after IR with 2 Gy. In case of HUVECs, the most pronounced effect on cell death was determined 24 h after IR, while the percentage of death cells in EAhy.926 and Hulec5a was the highest at 72 h and 96 h after IR, respectively. IR with 10 Gy caused significantly prolonged doubling times in all tested human EC lines, while a significantly prolonged doubling time after IR with lower doses was determined in EAhy.926 (4–8 Gy), and HUVEC (6 and 8 Gy) cell lines. To understand the dependence of EC gene expression on IR dose and time after IR, we compared RNA transcriptomic profiles of HUVECs at 24 and 72 h after 2 or 5 Gy IR with corresponding control (non-irradiated) samples. In addition, we investigated the temporal dynamics of HUVEC gene expression by comparing their transcriptomic profiles at 72 h and 24 h after IR with the same doses. Differential gene expression (DGE) analysis revealed a more pronounced effect of IR on HUVEC global gene expression at the dose of 5 Gy compared to 2 Gy. Furthermore, regardless of the dose, a greater effect of IR was observed at 72 h compared to 24 h after IR. A Functional Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis confirmed our results on the effect of IR on EC proliferation, showing, irrespective of the dose or time after IR, down-regulation of cell cycle progression-related signaling pathways and up-regulation of signaling pathways related to cell cycle arrest. To assess whether IR alone could lead to endothelial activation, the genes and pathways associated with it, including pathways related to changes in the extracellular matrix (ECM) were assessed. Compared to the control group, a significant up-regulation of Cell adhesion molecules, ECM-receptor interaction, Focal adhesion pathways, and Regulation of actin cytoskeleton were observed 24 h after IR with 2 Gy. Contrary, IR with 5 Gy did not induce similarly pronounced effect on ECM-related pathways in HUVECs, with only C-type lectin receptor signaling pathway being up-regulated at 24 h. Finally, to assess whether IR alone could lead to the changes in ECs favorable for the activation of the immune system, the genes and pathways specific for the activation of the immune response were examined. IR of HUVECs activated pathways related to the immune response at the transcriptional level. Regardless of the dose, significant up-regulation of the innate immune response, including NF-κβ signaling, TNFα signaling, and Cytokine-cytokine receptor interaction pathways, was observed at 24 h after IR. This suggests that immune response and pro-inflammatory pathways are activated in irradiated ECs as early as 24 h after IR. Additionally, pathways associated with the immune response remained significantly up-regulated at 72 h after IR. In order to establish an in vitro model that ensures the formation of perfusable 3D vasculature, thus provides a more physiological relevant model compared to monolayer-based assays, we have established a vasculature-on-a-chip model as a microfluidic assay that allows real-time monitoring of changes in the vasculature after IR. We evaluated two different vasculature-on-a-chip models.In the first model (Chen’s model), we established the optimal protocol, which resulted in perfusable microvasculature after 7 days of incubation as follows: 1.3 × 10⁴ HUVECs/μl mixed with NHLF in a 6:1 ratio in 5.5 mg/ml fibrinogen and 100 U/ml thrombin in EBM-2 medium. In addition, the microchips were incubated on a rocker with a 7° tilt changing every 8 min. To visualize the formed vasculature, we performed immunofluorescence staining for Vascular Endothelial cadherin (VE-Cad), VCAM-1, and VWF. However, the reproducibility of this model was low. Therefore, we tested an alternative model, OrganoPlate® microchips (Mimetas). Using this system, we successfully established reproducible vasculature-on-a-chip networks. The seeding and culturing procedure followed the manufacturer’s protocol, with the following changes: 1.0 × 10⁴ HUVECs were seeded and angiogenic factors were added daily from day 4 after seeding. On day 7 after seeding, we perfused the microchips with 70 kDa FITC labelled Dextran, to confirm that the formed vasculature networks were perfusable and not leaky. We then performed IR with either 2 or 5 Gy and followed changes in vascular permeability, morphology, and expression of endothelial activation markers VWF, ICAM-1, IRF9, and IFNα/β within the course of 72 h. The most pronounced effect on EC activation was 72 h after IR with 5 Gy, when we observed increased expression of VWF and ICAM-1, within the cytoplasm and membrane of ECs. This correlated with nuclear translocation of IRF9, characterizing its transcriptional activityand with an increased expression of IFNα/β, altogether indicating that activated endothelium exhibit a pro-inflammatory phenotype and contribute to the activation of immune response. To determine the level of vascular normalization, we analyzed the percentage of microchips growing perfusable, branched vascular networks. Results showed that IR did not lead to increased vascular normalization 24 h after IR, regardless of the dose. However, 72 h after IR, there was higher percentage of normal microchips after IR with 5 Gy, than in the control. This indicates that the vascular normalization induced by 5 Gy IR occurs at later timepoints. Interestingly, the percentage of perfusable microchips after 2 Gy was lower than the control suggesting that 2 Gy of irradiation does not have a vasculature normalizing potential. In contrast to vascular normalization, the number of CD8+ T cells interacting with ECs was significantly higher 24 h after IR with 2 or 5 Gy, in comparison to control. On the other hand, the number of CD8+ T cells interacting with ECs at 72 h after IR was comparable in irradiated versus non-irradiated microchips. In the second Work package, the effect of a single 15 Gy dose or a fractionated 5 × 5 Gy regimen of IR on tumor growth in CT26 and MC38 murine colon cancer models was evaluated in vivo. In both tumor models, a significant reduction in tumor volume was observed in irradiated tumors compared to controls, regardless of the IR regimen used. To assess whether and at which timepoint IR induces endothelial activation, fresh-frozen sections of irradiated and control colon carcinomas were stained for tumor endothelial cell (TEC) activation markers Vwf and Icam-1, as well as the TA-HEV marker Meca-79. IR induced a significant up-regulation of Vwf expression at all timepoints, regardless of the IR regimen, with the most pronounced effect observed 24 h after fractionated 5 × 5 Gy IR. Furthermore, fractionated IR dose caused significant up-regulation of Icam-1 and Meca-79, which were most elevated 24 h after IR. IR also caused up-regulation of Irf9, and increased infiltration of CD8+ and CD4+ T cells. The changes in hypoxia and the number of proliferative cells after IR were assessed using EdU and EF5 staining, respectively, showing IR-induced decrease of cell proliferation and hypoxia. In order to investigate dose- and time-dependent effect of IR on the transcriptome of irradiated murine CT26 colon tumors, within the spatial context of the TME, we then used the Visium Spatial Gene Expression platform (10X Genomics, US) to analyze FFPE sections of murine CT26 colon tumors. IR with fractionated 5x5 Gy dose led to up-regulation of TA-HEV associated genes in TECs at both 48 h and 7 days after IR. Additionally, we observed up-regulation of genes associated with immune response, which spatially colocalized with TA-HEV markers, suggesting an involvement of TA-HEVs and immune infiltration. This was then further confirmed at the protein level by immunohistochemical staining, with focus on regions with high TA-HEV transcript signatures. Despite different optimization parameters, we were not able to detect a Meca-79 positive TECs in any of the samples. However, immunohistochemical analysis confirmed increased expression of Irf9 and number of CD4+ and CD8+ T cells in irradiated tumors at both timepoints, supporting IR-induced immune activation. We next performed a spatial transcriptomic analysis on matched tumor biopsies from colorectal cancer patients taken before neoadjuvant radiotherapy (5×5 Gy) and samples from tumor resection taken after RT. The results showed that IR led to increased expression of TA-HEV-associated genes in TEC-positive regions. Consistent with in vivo experiments, IR led to up-regulation of immune-related genes that were spatially colocalized with TA-HEV markers. This was also confirmed on a protein level using immunohistochemical staining. In the third Work package, we determined how IR alters the homing and infiltration of immune cells, focusing on the interaction between TECs and CD8+ T cells, and the presence of TA-HEVs. In irradiated tumors, CD8+ T cells transmigrating across the vasculature and infiltrating into the TME were observed to a greater extent compared to control tumors. Next, we examined whether IR promotes the formation of TA-HEVs, by intravenously injecting anti-Meca-79 antibody into irradiated and control tumors growing in VE-TOM mice bearing dorsal skinfold window chambers. Although the Meca-79 signal was detected in one irradiated tumor, it was not observed in others. To validate the antibody delivery and functionality, lymph nodes from the same mice were harvested and imaged, showing Meca-79+ CD31+ vessels. This finding suggested that the low signal of Meca-79 observed in the tumors may be due to the limited abundance of Meca-79+ TA-HEVs or low expression levels of Meca-79 antigen on TECs, which made it impossible to perform intravital imaging of the interaction between CD8+ T cells and TA-HEVs.
In this doctoral dissertation, we elucidated how IR modulates tumor vasculature to support anti-tumor immunity. Specifically, using a state-of-the-art 3D vasculature-on-a-chip model and in vivo analyses, we demonstrated that IR,
Comparison of traditional and molecular methods of benthic invertebrate identification for the purpose of aquatic ecosystems quality assessment
No full textOcenjevanje stanja vodnih ekosistemov je eden izmed ključnih elementov za učinkovito upravljanje in varstvo teh ekosistemov. Pri monitoringu voda s pomočjo bentoških nevretenčarjev, kot kazalcev ekološkega stanja, se tradicionalno še vedno uporablja določanje na podlagi morfoloških značilnosti, ki zahteva veliko znanja in časa. Zato nas je zanimala primerjava z modernimi metodami sekvenciranja in njihova uporabnost v kontekstu monitoringa. Nabrali smo vzorce bentoških nevretenčarjev v litoralu Bohinjskega jezera in primerjali metodo določanja na podlagi morfoloških značilnosti z metabarkodiranjem vzorcev združbe nevretenčarjev in metabarkodiranjem tekočine, v kateri so bili nevretenčarji shranjeni (eDNA – okoljska DNA). Ugotovili smo, da pri računanju indeksa bentoških nevretenčarjev litorala jezer (LBI) z uporabo omenjenih metod pridemo do rezultatov, ki se statistično značilno ne razlikujejo. Z morfološkimi metodami ("morfo") smo zaznali več družin, z analizo DNA združbe nevretenčarjev ("bulk") in DNA shranjevalne tekočine ("eDNA") pa smo dosegli večjo taksonomsko natančnost. Časovno najbolj potratna metoda je bila "morfo", nato "bulk", najhitrejša pa "eDNA". Pregledali smo prednosti in pomanjkljivosti molekularnih metod.Ecological assessment of aquatic ecosystems is one of the key elements for their effective management and conservation. Morphological identification of benthic invertebrates as indicators of ecological status, is still used in monitoring programmes, requiring significant expertise and time. We were interested in comparing these methods with modern sequencing approaches and assessing their applicability in the context of monitoring. We collected samples of benthic invertebrates from the littoral of Lake Bohinj and compared the method of morphological identification with metabarcoding of bulk samples and preservative liquid (eDNA – environmental DNA). Our findings show that the differences between calculated Lake Biotic Indices (LBI) across the three methods are not statistically significant. We detected more invertebrate families using morphological identification but achieved higher taxonomic resolution using bulk or eDNA methods. Morphological identification was the most time consuming, followed by bulk sample metabarcoding, with eDNA metabarcoding being the fastest. Additionally advantages and disadvantages of molecular methods were reviewed
Development of an innovative, discrete in time, virtual sensor for proton exchange membrane fuel cell aging
No full textNizkotemperaturne gorivne celice s protonsko izmenjevalno membrano (PEMFC) so obetavna tehnologija za doseganje ničelnih strupenih emisij za uporabo v težkih tovornih vozilih. Kljub napredku na področju zmogljivosti, učinkovitosti in življenjske dobe komponentna degradacija zaradi obratovalnih režimov ostaja izziv, vreden naslovitve. V magistrski nalogi se osredotočamo na analiziranje zmogljivosti PEMFC na različnih točkah življenjske dobe in razvoja metodologije za uporabo modela kot virtualnega zaznavala za spremljanje stanja staranja komponent PEMFC. Na podlagi eksperimentalnih podatkov, v katerih je zajeto spreminjanje zmogljivosti PEMFC skozi celotno življenjsko dobo za različne materiale katalizatorja, smo z razvito metodologijo diskretnega virtualnega zaznavanja staranja PEMFC kalibrirali elektrokemijski model in spremljali evolucijo pridobljenih intrinzičnih parametrov. Model na validacijskih podatkih izkazuje dobro napovedljivost, saj dosega visoko stopnjo ujemanja z eksperimentalnimi podatki z najnižjo R2 vrednostjo 0,96709, ob sočasni unikatni določljivosti vseh parametrov, ki je bila ocenjena s statistično analizo Fisherjeve informacijske matrike. Slednja nam omogoča tudi edinstven uvid v količino informacije, ki jo nosi individualna eksperimentalna točka. Rezultati kažejo, da razvita metodologija
za diskretno virtualno zaznavo staranja PEMFC ustrezno zazna degradacijo treh intrinzičnih parametrov, potrjenih s primerjavo z in-situ meritvami elektrokemijsko aktivne površine, visoko frekvenčne upornosti, napetosti in toka ter ex-situ meritvami
poroznosti, debeline membrane in slikovnega gradiva.Low temperature proton exchange membrane fuel cells (PEMFC) are a promising solution to achieve zero toxic emissions in heavy duty vehicles. Despite progress in performance, efficiency and envisaged lifetime, component degradation under operating
conditions remains a key challenge. This work analyses PEMFC performance at different points of lifespan and develops a methodology to use a model as a virtual sensor to monitor component degradation in a PEMFC. Using experimental data that envelops performance measurements across the entire PEMFC lifespan for different catalyst materials, we calibrated the electrochemical model using the developed methodology of discrete virtual sensing of PEMFC aging and noted the evolution of its intrinsic parameters. The model exhibits good predictability, achieving a high degree of agreement with experimental data with the lowest value of R2 being 0,96709, while simultaneously obtaining uniquely identifiable parameters, confirmed by Fisher information matrix analysis. It also enables a unique view into the amount of information provided from each experimental point. Results show that the proposed virtual sensing methodology successfully detects degradation of three intrinsic parameters, confirmed by comparison with in-situ measurements of the electrochemically active surface area, high frequency resistance, voltage, current and ex-situ measurements of porosity, membrane thickness and other imaging material